The first inhibitor of human DHOD was reported in 1985, and NSC368390 (renamed later as Brequinar) caused 90% or more inhibition of the growth of human MX-1 breast, LX-1 lung, BL/STX-1 stomach and CX-1 colon carcinomas in nude mice at a dose of 20 to 40 mg/kg/day, and was also effective against HCT-15, clone A and DLD-2 tumors, particularly its growth inhibition of DLD-2 colon cancer was as high as 98% (Non-patent Document 1). In the next year, UMP levels in NSC368390-treated cells were found to be reduced by 50% after treatment with 25 μM NSC368390, and isotope-labeled substrates were used to measure the activity of all enzymes responsible for the pyrimidine de novo synthetic pathway, indicating that the fourth enzyme DHOD was strongly inhibited (Ki=23.5 nM) (Non-patent Document 2). In 1989 (USA) and in 1990 (Netherlands), Phase I clinical trials were initiated, and 45 and 43 solid tumor patients, respectively, were administered one after another. The results obtained are reported in Non-patent Document 3 and Non-patent Document 4. In the same year. Brequinar was reported to be more effective than five drugs used for treatment of head and neck squamous cell carcinomas.
In 1993, experiments in mice showed that allograft rejection was caused later by Brequinar derivatives, suggesting that DHOD inhibitors have an immunosuppressive effect. In 1994. Brequinar was reported to suppress the growth of lymphocytes and was also reported to inhibit cell cycle transition from the G0/G1 phase to the S and G2+M phases (Non-patent Document 5). In 1995, there was a report showing that the target of Leflunomide used for treatment of rheumatoid arthritis was human dihydroorotic acid dehydrogenase (hereinafter abbreviated as HsDHOD); and hence DHOD was confirmed again to be promising as a target for anticancer and immunosuppressive purposes, as in the case of Brequinar. DHOD purified from mouse spleens was identified to bind to A771726 (Leflunomide metabolite) with high affinity, thus indicating that Leflunomide is metabolized in vivo into A771726, and this A771726, but not Leflunomide, has an immunosuppressive effect (Non-patent Document 6, Non-patent Document 7). It should be noted that A771726 binds to the ubiquinone-binding site in known HsDHOD inhibitors. In 1996, NSC 665564 was reported to inhibit HsDHOD at the same level as Brequinar and thereby suppress the growth of various cancer cells (Non-patent Document 8). In 1998, Leflunomide was reported to arrest the growth of T-lymphocytes in the G1 phase. In the same year, esters of Leflunomide were found to suppress the growth of B-lymphocytes and hence were proposed as therapeutic agents for graft rejection in organ transplantation.
In 2000, A771726 suppressed diabetic symptoms in a concentration-dependent manner in the NOD (non-obese diabetic) mouse model of diabetes, and hence inhibitors of human DHOD were found to be effective in T cell triggered disease, i.e., insulin-dependent diabetes mellitus (IDDM).
The following reports have been issued for DHOD in non-human organisms. Membrane-bound DHOD is used as a drug target for malaria (Non-patent Document 9, Non-patent Document 10) and also for Helicobacter pylori (Non-patent Document 11, Non-patent Document 12) and Candida albicans (Non-patent Document 13, Non-patent Document 14).
In addition, an analog (FK778) of Leflunomide has already been reported to suppress the growth of human cytomegalovirus (Non-patent Document 15). Moreover, Brequinar has been reported to suppress the growth of flaviviruses (dengue virus, West Nile virus, yellow fever virus, and Powassan virus), plus-strand RNA alphavirus (Western equine encephalitis virus) and negative-strand RNA rhabdovirus (vesicular stomatitis virus) (Non-patent Document 16). At last, in March 2011, it was reported in PNAS (Proc Natl Acad Sci USA) that a compound (Compound A3) suppressing the growth of a wide range of viruses [negative-sense RNA viruses (influenza viruses A and B, Newcastle disease virus, and vesicular stomatitis virus), positive-sense RNA viruses (Sindbis virus, hepatitis C virus, West Nile virus, and dengue virus), DNA viruses (vaccinia virus and human adenovirus) and retroviruses (HIV)] was found through HTS (high-throughput screening) (Non-patent Document 17). The inhibitory effect of this compound on virus growth was suppressed by orotic acid, but not suppressed by dihydroorotic acid which is a substrate of DHOD, thus indicating that the target of Compound A3 inhibits dihydroorotic acid dehydrogenase present in human mitochondria and thereby exerts an inhibitory effect on virus growth (Non-patent Document 17). Further, as a result of analyzing their toxicity on human cells, these compounds were found to show 1500-fold to 2400-fold or more selectivity between cytotoxicity (CC50) and virus growth inhibition (IC50) depending on the type of cells. The reason that these compounds have an inhibitory effect on virus growth has been confirmed to be because viral RNA-DNA synthesis is stopped upon reduction of the pyrimidine pool in the infected cells (Non-patent Documents 19 to 21).
As described above, in humans. DHOD inhibitors have been known to be promising as anticancer agents or immunosuppressive agents since 1980s, and studies are also actively proceeding now. As a mechanism for immunosuppression, DHOD inhibitors suppress the growth of activated T-lymphocytes and B-lymphocytes. The pyrimidine pool in normal cells is mediated by uracil transport, salvage pathway and de novo synthesis. However, activated lymphocytes and cancer cells depend on de novo synthesis. Since 1990s, it has been reported that upon inhibition of DHOD, normal cells can survive due to the uracil transport and salvage pathway, whereas lymphocytes and cancer cells cannot grow.
Moreover, DHOD inhibitors are suggested as drug targets for T-lymphocyte-mediated diabetes.
In the other organisms, membrane-bound DHOD is known as a drug target in malaria and H. pylori, and many articles have been reported for drug design using DHOD as a target. On the other hand, DHOD inhibitors are known to be imperative for candidiasis because they exert growth inhibition.